AN AUTORADIOGRAPHICAL STUDY OF THE LOCALIZATION OF THE UPTAKE OF GLUTAMATE BY THE PERIPHERAL NERVES OF THE CRAB, CARCINUS MAENAS (L.

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1 J. Cell Sci. 14, (i974) 351 Printed in Great Britain AN AUTORADIOGRAPHICAL STUDY OF THE LOCALIZATION OF THE UPTAKE OF GLUTAMATE BY THE PERIPHERAL NERVES OF THE CRAB, CARCINUS MAENAS (L.) P. D. EVANS* Department of Zoology, University of Cambridge, Downing Street, Cambridge, England SUMMARY An autoradiographical localization of the uptake sites of [ 3 H]glutamate has been performed on the peripheral nerves of Carcinus maenas (L.) at both the light- and electron-microscope levels. Biochemical control analyses have also been carried out at various stages of the fixation procedure. A statistical analysis of the grain distribution between the glial (and connective tissue) and axonal compartments revealed that it was highly non-random. A comparison has been made of the grain distribution obtained with the ' circle analysis' method with that from the 'hypothetical grain distribution analysis'. The latter analysis revealed that the specific activity of label in the glial compartment was about seven times that of the axonal compartment. The significance of a preferential accumulation of glutamate by the glial compartments in this tissue is discussed in relation to similar findings in other tissues. INTRODUCTION The uptake of L-glutamate by isolated peripheral nerve bundles from the walking leg of the common shore crab, Carcinus maenas, has been described in terms of its kinetics, specificity and energy sources (Evans, 1973 a). However, ultrastructural studies on this tissue, such as those by Horridge & Chapman (1964), Baker (1965) and the present study, have revealed that the nerve bundle is composed of a heterogeneous population of cells, including glial cells and axons of varying sizes. Thus the results of experiments on the uptake and efflux of radioactive tracers are difficult to interpret in terms of the contribution of each of the cellular compartments to the overall observed effect. The present study describes the autoradiographical localization of accumulated radioactively labelled glutamate at the level of both the light and electron microscopes. The amino acid binding artifact of glutaraldehyde fixation (Peters & Ashley, 1967) has been used to locate the sites of the amino acid accumulation in a manner similar to that used in previous studies of glutamate uptake into a cockroach nerve-muscle preparation (Faeder & Salpeter, 1970, and Salpeter & Faeder, 1971) and of y-aminobutyric acid (GABA) uptake in a lobster nerve-muscle preparation (Orkand & Kravitz, 1971), rat brain (Bloom & Iversen, 1971) and cockroach brain (Frontali & Pierantoni, Present address: Harvard Medical School, Department of Neurobiology, 25 Shattuck Street, Boston, Mass , U.S.A.

2 352 P. D. Evans 1973). It was of interest to see if this technique when applied to crab peripheral nerve would reveal any differences in the uptake between the different cell types in the bundles of small axons with their thin glial sheaths and in the large motor axons with their layered glial and connective tissue investments. Biochemical control analyses of the distribution of radioactivity between the soluble and insoluble fractions of nerve extracts have been carried out at various stages of the fixation procedure. The results of such studies are discussed together with other difficulties in the interpretation of data obtained from autoradiographic localization studies of water-soluble molecules. MATERIALS AND METHODS Incubation conditions Peripheral nerves from the walking legs of Carcinus were dissected out in saline as described by Evans (19736). Small pieces of nerve were ligatured with hairs at both ends and immersed in 5 ml of saline containing DL-[*H]glutamate (sp. act. 3-8 Ci/mmol, Radiochemical Centre, Amer8ham) at a final concentration of 26 x io~ a miw and 100 /ici/ml. They were incubated at 18 C with shaking for 1 h. After incubation they were shaken in fresh unlabelled saline medium for 2 periods of 10 min each at o C, to wash the radioactivity out of the extracellular spaces. Fixation The fixation and embedding procedures (Dr B. L. Gupta, personal communication) used were essentially the same for both light and electron microscopy. After incubation and washing, the short pieces of nerve were fixed for 3 h in 5 % glutaraldehyde in o-i M cacodylate buffer plus 400 mm sucrose, adjusted to ph 7-1. They were then rinsed in an isotonic buffer wash for three 45-min periods prior to postfixation for 1 h in a 1 % isotonic solution of osmium tetroxide. The samples were then rinsed briefly, dehydrated in an ethanol series and embedded in Araldite (Ciba, England). Autoradiography The methods used in the present study were derived from the procedures developed by Salpeter and Bachman (see Salpeter & Bachmann, 1964; Bachmann & Salpeter, 1967; and Faeder & Salpeter, 1970). Light microscopy. Thick sections (0-5-1-o fim) were cut using a Huxley ultramicrotome with glass knives and the sections mounted upon clean glass slides. They were then dipped into melted Ilford L4 Nuclear Research emulsion (Ilford, Ltd., Ilford, Essex) diluted 1:2 with triple-distilled water containing % lauryl sulphate (Moses, 1964) at a temperature of 35 C C, dried and kept in light-tight boxes at 4 C. Sets of slides were developed at intervals over a 1-4 week period using Kodak D19 and fixed in 25 % sodium thiosulphate. Some of the sections were then stained with 1 % methylene blue in 1 % sodium tetraborate solution for 10 s and were examined under bright-field or phase-contrast illumination on a Zeiss Universal microscope and photomicrographs were taken using a Wild Micro-photoautomat. The relative specific activities of label in each of the cellular compartments were estimated by comparing the percentage of grains occurring over each cell type with a measure of the relative areas occupied by each cell type from a point analysis of the same fields. Electron microscopy. Sections showing silver to pale gold interference colours were dried on to slides coated with o-8-i-o% Celloidin (ICI) in isoamyl acetate. The sections were stained with uranyl acetate (saturated solution in 50% ethanol) and lead citrate (Reynolds, 1963). After thorough drying an approximately 50-nm thick carbon film was evaporated on the surface bearing the sections. The slides were then dipped using a semi-automatic dipping device (Kopriwa, 1966; Vrensen, 1970) into melted Ilford L4 emulsion diluted 1:2-5 with distilled

3 Glutamate uptake by crab peripheral nerve 353 water as described above. They were dried and stored as above. Autoradiographs were developed after a week exposure using Kodak Dit)b and fixed in 25 % sodium thioaulphate. The Celloidin films were stripped from the slides by flotation on to distilled water and the tissue sections were picked up on to copper grids. The autoradiographs were examined in a Philips EM 300. Areas were photographed at a constant magnification (x ) and printed x 3 to give a final magnification of x The distribution of the silver grains was then examined using 2 analytical procedures. In the first the 'circle analysis' of Williams (1969) was used; the results of this were compared with those obtained from the application of the recently described analysis using hypothetical grain distributions (Blackett & Parry, 1973; and Parry & Blackett, 1973). In the former analysis the centre of each grain was located and used to draw a circle equal to the ' resolution' of the system used (i.e. a circle of 50 % confidence of enclosing the site of disintegration) (Salpeter, Bachmann & Salpeter, 1969). The circle has a radius of approximately 250 nm (equivalent to 7-5 mm radius on diameter electron micrographs) for Ilford L4 emulsion, assuming a section thickness of 50 nm. The grain circles were then assigned to either axonal, glial (and connective tissue) or junctional compartments. The effective areas occupied by each of the above compartments were calculated using circles (of same diameter as above) arranged randomly on a transparent screen. A Chi-squared statistical comparison between the circles and grain frequency distribution was performed to examine whether or not the grain distribution was random (Williams, 1969). The number of silver grains per unit area of a cellular compartment can be used as a measure of the radioactivity per unit volume. Thus a comparison of the percentage grain frequency with the percentage effective area occupied by each compartment from the same micrographs enables a relative specific activity to be obtained for each compartment (Williams, 1969). In the second analysis a set of computer-generated random distances and directions (kindly supplied by Drs Blackett & Parry) was suitably modified to the conditions uaed in the present study (Blackett & Parry, 1973), and used to generate a series of hypothetical grain densities in the various cellular compartments. The circles used had the same diameter as in the previous analysis. The hypothetical distribution was then compared with the real distribution of grains and the activities in the various structures altered until a statistically good fit was obtained using the Chi-squared test of significance. Control analyses Pieces of nerve were incubated as described above in either the above DL-['H]glutamate or a L-[ 14 C]glutamate (sp. act. 260 mci/mmol, Radiochemical Centre, Amersham, 1 /ici/ml and 004 HIM) containing saline for periods of 20 min. They were then homogenized in 60 % aqueous ethanol and the distribution of the radioactivity determined between the soluble and insoluble fractions as described previously (Evans, 1973 a). This procedure was repeated at different stages of the fixation schedule described above. An examination of the effect of various proteinsynthesis inhibitors such as io~* M cycloheximide, puromycin and chloramphenicol on the distribution of the activity between the 2 fractions was also carried out. RESULTS Autoradiography The phase-contrast micrographs shown in Figs. 1-4 are the result of a 4-week exposure. Figs. 1 and 2 show the distribution of silver grains over typical regions of tissue containing small and medium-sized axons, whilst Figs. 3 and 4 show the grain distribution over typical transverse sections of giant axons with their complex sheath structures. It can be seen from the above micrographs that grains appear over both glial and neuronal regions. Table 1 shows the distribution of total activity in terms of grain density, between the various cellular compartments of this tissue estimated over a

4 354 P- D- Evans large number of sections (n = 25). The statistical analysis indicated that the grains were distributed in a highly non-random manner. The relative specific activities (i.e. percentage silver grains/percentage area) of label in the glial and connective tissue compartments were about 3 times that of the axonal compartment. Table 1. Distribution of silver grains appearing over glial and axonal compartments based on examination of light-microscope autoradiographs The figures in parentheses represent the relative areas of the compartments from a random point analysis X* = 395, P < oooi, i.e. grain distribution is highly nonrandom. The data suggest that the relative specific activity of label in the glial and connective tissue components is about 3 times that of the axons. % grains appearing over Glia Axons Giant axons (32-3) (677) Bundles of medium and small axons (26-3) (73-7) Total (28-3) (71-7) Table 2. Distribution of silver grains appearing over glial {and connective tissue), axonal and junctional compartments based on 20-weeA electron-microscope autoradiographs. The random circle analysis was performed as described by Williams (1969) Axon Glia/connective tissue Junctional A/G Total No. of grains observed (0) No. of random circles Grains expected (E) x* = (O-E) 2 /E Probability OOOI o-oi o-oi '4 o-ooi The results obtained with an electron-microscope autoradiographical study were essentially the same after both 10- and 20-week exposures. The data presented in this paper come from a 20-week exposure. Figs. 5 and 6 show electron-microscope autoradiographs of typical regions containing bundles of small and medium-sized axons, whilst Figs. 7-9 show regions containing giant axons. Table 2 contains the results of a circle analysis (Williams, 1969), which reveals that the distribution of the grains was again highly non-random. An estimate of the relative specific activity of the label in the glial (and connective tissue) compartment with respect to the axonal compartment can be made by distributing the junctional grains and circles as described by Williams (1969). This reveals that 52-4 and 47-6% of the total activity is found in the glial and axonal compartments respectively and also that they occupy 31-3 and 687 % of the area of the tissue respectively. This suggests that

5 Glutamate uptake by crab peripheral nerve 355 the relative specific activity of label in the glial (and connective tissue) compartment was about 2-4 times that of the axonal compartment. This rinding was in close agreement with that from the light-microscope study. Table 3. Hypothetical grains for chosen activity throughout sections Site of hypothetical grains Source of hypothetical grain VJ ruins per unit area Axon 1 Glia/connective tissue 1 Total Real grains Expected t P t Axon Glia/connective tissue Junctional A/G i Total Activity ratio axonal/ glial components Whole nerve bundle Real grains : 1 :2 :3 = 4 5 :6 7 :8 :9 : i i Total X i Bundles of small and mediumsized axons Real grains 1 :8 9 : 10 : 11 : 12 1 :i The second analysis, along the lines described by Blackett & Parry (1973) (see Table 3) again revealed that the distribution of grains was highly non-random. However, it revealed that the best fit to the experimental data was obtained when the axonal/glial specific activity ratio was about 1:7 when the analysis was performed on the whole nerve bundle. When the analysis was confined to the small and medium-sized bundles, excluding the giant motor axons, the best fit ratio was even higher at 1:10. The significance of the differences between the 2 methods of analysis is discussed later.

6 356 P. D. Evans Biochemical controls The distribution of label between the soluble and insoluble fractions of a 60% aqueous ethanol extract of peripheral nerve from Carcinus is shown in Table 4. It can be seen that for both isotopes used there was a very low incorporation of label into the insoluble fraction measured after the post-incubation wash. However, after the 3-h fixation in 5 - o% glutaraldehyde and the three 45-min buffer washes, the percentage of the label in the insoluble fraction increased. This was presumably due to the amino-acid binding artifact of glutaraldehyde fixation described by Peters & Ashley (1967). The corresponding percentages of label in the soluble fraction were reduced by loss to the medium. In the case of L-[ 14 C]glutamate only 17% of the total activity was left in the tissue after the buffer wash but the corresponding percentage for DL-[ 3 H]glutamate was 30%. Table 4. Distribution of label between soluble and insoluble fractions of 60 % aqueous ethanol extraction at various stages of fixation procedure for 14 C- and 3 H-labelled glutamate The results are expressed as a percentage of the total radioactivity found in the nerve after the post-incubation wash which removed the incubation medium from the extracellular space. Stage After post-incubation wash After postfixation buffer wash % label in 60 % aqueous ethanol fractions Isotope ^ Soluble Insoluble 14 C »H U C H 2O-O 16-0 It would thus appear that the incorporation of label into the insoluble fraction prior to fixation could introduce a source of error in the estimation of the distribution of soluble activity by autoradiographic means. It can be seen that the size of the component increases during the fixation procedure as more of the soluble component is washed out of the tissue. Attempts were therefore made to reduce this source of error to a minimum by the use of standard protein synthesis inhibitors. Experiments have been performed using the inhibitors cycloheximide, puromycin and chloroamphenicol, all at io~ 3 M, both separately and in various combinations. At present the results are somewhat equivocal and no combination has been found which will completely abolish the incorporation into the 60% aqueous ethanol-insoluble fraction after the radioactive incubation prior to fixation. It could be that other processes beside protein synthesis are causing binding of label to the insoluble fraction. Experiments are continuing on this problem to minimize its effect on the interpretation of autoradiographic data for the distribution of soluble free amino acid molecules.

7 Glutamate uptake by crab peripheral nerve 357 DISCUSSION The interpretation of data obtained in an autoradiographic analysis of the distribution of radioactively labelled water-soluble amino acid molecules not incorporated into protein in a tissue is fraught with difficulties. Possible artifacts could originate from protein synthesis (which could be more pronounced in one cellular compartment than another), from the movement of label from one compartment to another during incubation and fixation, from the irreversible binding of the label to protein molecules, from the lack of knowledge of the degree of selective washout from specific structural compartments and from the metabolism of the label itself. In the present study controls have been performed to estimate the likely size of errors due to protein incorporation of the label and further inhibitor studies are at present in progress to attempt to minimize this source of error. There seems to be little evidence for any selective washout phenomenon from the proteinaceous compartment of a particular structural compartment during fixation (Dr B. L. Gupta, personal communication). Also Orkand & Kravitz (1971) have shown that tritiated y-amino-butyric acid (GABA) added to the medium immediately after fixation in a non-radioactive medium produced very little binding. This suggested that the GABA in their experiments was bound only at the sites where it was localized prior to fixation and that no artifacts due to the leaching of label from one compartment to another could be detected during fixation. The latter possibility would have involved the GABA crossing cell membranes during fixation (when presumably transport was inactivated). It has also been shown that L-glutamate taken up by the peripheral nerves of Carcinus is metabolized to other soluble products at only a low rate (Evans, 1973 a). The present studies at both the light- and electron-microscope levels have indicated that label was taken up into both the glial and neuronal structural compartments. It appeared from a survey of the grain distribution that the total activity in the tissue was fairly evenly divided between these compartments. This tends to support the hypothesis that the fast and slow components of the radioactive efflux from the peripheral nerves of Carcinus loaded in saline containing labelled glutamate (Evans, 1973 c) might be identified as the effluxes from the glial and neuronal compartments respectively, the rapid efflux possibly originating from the thin interdigitating folds of the glial cells around the axons, and the slower phase from the axons themselves. Both the statistical analyses of the grain distribution revealed that the grains were distributed in a highly non-random manner. However, the Blackett and Parry analysis suggested the glial (and connective tissue): axonal specific activity ratio might be as high as 7:1, whereas the Williams analysis gave a ratio of only 2-4:1. The former analysis has the advantage of including the effects of cross-fire between the various compartments, since it relates to the particular shapes and sizes of the structures observed in the autoradiographs and also provides estimates for the activity within different structures consistent with the distribution of the real autoradiographic grains. Thus it can be seen that the true extent of the preferential accumulation of radioactively labelled glutamate by the glial (and connective tissue) compartment is not realized with the Williams analysis alone.

8 358 P.D. Evans If the Blackett and Parry analysis is confined to the relative specific activity distributions in areas containing only medium and small-sized axons, then the present data indicate a different best fit ratio to that found for the whole tissue. This suggests that the relative specific activity ratio for glutamate uptake might be different in the bundles of small and medium sized axons when compared to the giant axons. This result could be explained by the uptake of glutamate being more effective either in the glial compartment of the small axons or in the axonal component of the giant axons. However, in view of the fact that the Na+-dependent uptake system for glutamate in this tissue seems to be monophasic (the Na + -independent system being negligible, < i % of total, at the low glutamate concentration of the incubation medium) (Evans, 1973 a), it seems likely that the preferential uptake by the glial system, as well as perhaps the difference between the relative specific activity distribution ratio between the giant axons and the bundles of smaller axons, might both be accounted for in terms of the larger surface-to-volume ratio of the glial compartment compared with the axonal compartment. A preferential uptake of amino acid by glial cells has also been reported for glutamate in a cockroach nerve-muscle preparation (Faeder & Salpeter, 1970) and for GAB A in a lobster nerve-muscle preparation (Orkand & Kravitz, 1971). These results have been interpreted as indicating a possible means of protecting the neuromuscular junctions against blood amino acids, as a means of conservation of the amino acids released as transmitters, as well as a possible mode of transmitter inactivation. The finding in the present study that the glial cells of the peripheral nerve bundle also accumulate glutamate preferentially indicates that this phenomenon is not restricted to the glial cells in the immediate vicinity of the neuromuscular junction, but is rather a general property of glial cells. I would like to thank Drs B. L. Gupta and N. J. Lane-Perham for their helpful advice and instruction in the techniques of autoradiography and electron microscopy, and also for reading the manuscript. I would also like to thank Dr J. E. Treheme for his helpful discussions during the course of this work and Dr D. M. Parry for kindly supplying the set of computer-generated random distances and directions. The work was carried out during the tenure of an S.R.C. Research Studentship. REFERENCES BACHMANN, L. & SALPETER, M. M. (1967). Absolute sensitivity of electron microscope radioautography. J. Cell Biol. 33, BAKER, P. F. (1965). A method for the location of extracellular space in crab nerve..7. Physiol., Lond. 1, BLACKETT, N. M. & PARKY, D. M. (1973). A new method for analysing electron microscope autoradiographs using hypothetical grain distributions. _?. Cell Biol. 57, BLOOM, F. E. & IVERSEN, L. L. (1971). Localisation of 'H-GABA by electron microscope autoradiography. Nature, Lond. 329, EVANS, P. D. (1973 a). The uptake of L-glutamate by the peripheral nerves of the crab Carcimis maenas (L.). Biochim. biophys. Ada 311, EVANS, P. D. (1973ft). Amino acid distribution in the nervous system of the crab Carcimis maenas (L,.).J. Neurochem. 21, EVANS, P. D. (1973 c). The stability of the free amino acid pool in isolated peripheral nerves of Carcinus maenas (L.)._7. exp. Biol. 59,

9 Glutamate uptake by crab peripheral nerve 359 FAEDER, I. R. & SALPETER, M. M. (1970). Glutamate uptake by a stimulated insect nervemuscle preparation. J. Cell Biol. 46, FRONTALI, N. & PIERANTONI, R. (1973). Autoradiographic localization of 3 H-GABA in the cockroach brain. Comp. Biochem. Physiol. 44 A, HORRIDCE, G. A. & CHAPMAN, R. A. (1964). Sheaths of the motor axons of the crab Carcimts. Q.Jlmicrosc. Set. 105, KOPRIWA, B. M. (1966). A semi-automatic instrument for the radioautographic coating technique.^. Histocliem. Cytochem. 14, MOSES, M. J. (1964). Application of autoradiography to electron microscopy. J. Histochem. Cytochem. 12, ORKAND, P. M. & KRAVITZ, E. A. (1971). Localization of the sites of GABA uptake in lobster nerve-muscle preparations.^. Cell Biol. 49, PARRY, D. M. & BLACKETT, N. M. (1973). Electron microscope autoradiography of erythroid cells using radioactive xron.j. Cell Biol. 57, PETERS, T. & ASHLEY, C. A. (1967). An artifact in radioautography due to binding of free amino acids to tissues by fixatives.,?. Cell Biol. 33, REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SALPETER, M. M. & BACHMANN, L. (1964). Autoradiography with the electron microscope. J. CM Biol. 22, SALPETER, M. M., BACHMANN, L. & SALPETER, E. E. (1969). Resolution in electron microscope autoradiography. J. Cell Biol. 41, SALPETER, M. M. & FAEDER, I. R. (1971). The role of sheath cells in glutamate uptake by insect nerve muscle preparation. Prog. Brain Res. 34, VRENSEN, G. F. J. M. (1970). Some new aspects of efficiency of electron microscope autoradiography with tritium. J. Histocheni. Cytochem. 18, WILLIAMS, M. A. (1969). The assessment of electron micrographs. In Advances in Optical and Electron Microscopy, vol. 3 (ed. R. Barer & V. E. Cosslett), pp New York and London: Academic Press. (Received 13 June 1973)

10 360 P. D. Evans Figs. 1, 2. Light-microscope autoradiographs developed after a 4-week exposure and photographed under phase-contrast illumination with the focus on the silver grains lying above the tissue. The figures show the distribution of silver grains over typical regions of the nerve bundle containing small and medium-sized axons (a). It can be seen that grains occur over both the glial and connective tissue regions as well as over the axonal regions, b, boundary of nerve bundle, x 2400.

11 Glutamate uptake by crab peripheral nerve

12 362 P. D. Evans Figs. 3, 4. The figures show the distribution of grains over typical transverse sections of 'giant' axons with their complicated sheath structure of layered glial processes and connective tissue. The conditions were as for the preceding figures. It can be seen that silver grains occur over both the sheath regions (sh) of these axons and over the centres of the axons themselves (a), x 2400.

13 Glutamate uptake by crab peripheral nerve 363 C E I. 14

14 364 P. D. Evans Figs, s, 6. Electron-microscope autoradiographs developed after a 20-week exposure showing typical regions from the peripheral nerve bundle containing small and medium-sized axons. Silver grains lie above both the axonal bodies (a) and the thin glial processes (gl) and connective tissue (e) regions between the axons. x

15 Glutamate uptake by crab peripheral nerve v r. F 36s

16 366 P. D. Evans Figs. 7, 8. Electron-microscope autoradiographs developed after a 20-week exposure showing typical regions of peripheral nerve bundle containing 'giant' axons with their complex glial and connective tissue sheaths. Silver grains lie above both the axonal bodies and over the sheath regions (sh). The sheath region can be seen to consist of an inner region of interdigitating pavement glial cells (t) about 1 /tm thick and an outer region of alternating glial cell processes and layers of extracellular fibrillar material in a dense matrix, x Fig. 9. A higher-power electron-microscope autoradiograph prepared as above showing the sheath region between 2 giant axons (a). Silver grains can be seen to be associated with both the inner pavement glial cells (i) and the region containing the alternating layers of glial processes and extracellular fibrillar material, x

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